Field of the Invention
The present invention relates to a detection system of a test substance using a magnetic body and a marker.
Description of Related Art
An ELISA (enzyme-linked immunosorbent assay) method, an MEIA (microparticle enzyme-based immunoassay) method, and the like have been widely used as the method for detecting a small amount of test substance in a sample. However, there are problems that these methods require a long time for operation or reaction, and include complicated measuring operation.
In recent years, an analytic method using immunochromatography has been attracting attention as alternative analytic method to the ELISA method or the like. The immunochromatography is an immunoassay that determines the presence or absence of a test substance. Specifically, the test substance moves in a porous support by a capillary phenomenon, and it is captured by a marker. Then, the test substance is concentrated by contact with a capturing substance locally (for example, a linear shape) immobilized with the porous support. Then, the place where the capturing substance is immobilized is colored.
The immunochromatography is excellent in the light of stable storage, quick measurement, easy determination, and no particular device. Thus, the immunochromatography has been attracting attention as a novel POCT (point of care testing) technique and is used for a pregnancy test and an influenza test
In some cases, however, there are cases that the immunochromatography cannot show sufficient sensitivity or cannot detect any markers in some analytical targets. In current immunochromatography, gold nanoparticles have been mostly used as marking substances. However, if the test substance in a sample is extremely low concentration, an erroneous decision to be negative is often encountered by very faint color development due to the insufficient amount of gold nanoparticles accumulated in the test line.
Therefore, researches designed to improve the detection sensitivity of the immunochromatography are being investigated in various research fields.
Patent Document 1 (Japanese Patent No. 4514824) discloses a method of binding of an antibody that recognizes a test substance to a silica nanoparticle containing a fluorescent substance and detection of fluorescence of the silica nanoparticle bound to the test substance.
Patent Document 2 (Japanese Patent No. 4179419) discloses a method of increase of the accumulated amount of a test substance and acquirement of a signal of the test substance by developing a first antibody-bound sensitizer which emits a given signal after development of the test substance and a marking second antibody that recognizes the test substance in the first antibody-immobilized development medium, in the case of low concentration of the test substance.
Patent Document 3 (Japanese Patent No. 4791867) discloses a method of detection of a test substance in short time by binding a first antibody to a magnetic body coated with a noble metal material and by accumulating a complex composed of the test substance and the first antibody-bound magnetic body on a region where a second antibody is immobilized in a development medium using a magnetic force.
Patent Document 4 (Japanese Unexamined Patent Application, First Publication No. H5-52849) discloses a method of specific detection of a substance to be measured, by reacting the substance to be measured in a sample with a substance having a magnetic property and specifically reacting with the substance to be measured, and with a marked substance specifically reacting with the substance to be measured in a position different from that of the substance having a magnetic property, by causing a marked reaction product which has a magnetic property to move by capillary movement, and by capturing the reaction product at a specific position of a capillary movement medium using a magnetic force.
However, Patent Document 1 discloses the method that in the case of low concentration of the test substance, the reaction efficiency between the test substance and the silica nanoparticle decreases, and then, the test substance may be erroneously determined to be negative.
In addition, Patent Document 2 discloses the method that the developing operation and the detecting operation are complicated, and therefore, a long time may be required until the detection result is obtained, due to complicated operations in development and detection.
In addition, Patent Document 3 discloses the method that since a magnetic body which do not form complex with the test substances is accumulated, a cleaning operation may be required and detection of the test substance may be erroneously determined due to the particles which cannot be removed. In addition, Patent Document 4 discloses the method that uses blended yarn fabric of nylon and tetron as a chromatography medium. However, use of such a chromatography medium slows the developing speed of the test substance and the magnetic body which are added to the chromatography medium. In addition, when a color development reaction occurring in the chromatography medium is detected from the outside of the chromatography medium, sufficient sensitivity may not be obtained due to blocking by the chromatography medium.
In the case of the above-described immunochromatography, immobilization of an antibody or an antigen (mostly antibody), which is separately from a marked antibody or antigen and recognizes a test substance, on a development medium is required. Therefore, the development medium that has characteristics not only suitable for capillary development of a sample but also suitable for immobilization of an antibody or an antigen is required. Moreover, there are stringent requirements for the material of the development medium.
Furthermore, in the above-described immunochromatography, the reaction between a test substance and an antibody which is immobilized on the development medium or between a test substance and a marked antibody occurs instantly in the capillary development. Therefore, in order to detect the test substance with high sensitivity, immobilization of an antibody with high binding constant on the development medium is required. The types of antibodies or antigens for immobilization are restricted.